Generally, enzymes are water soluble. Thus, when utilized in a reaction medium without being immobilized on a support (free enzyme), they are difficult to remove therefrom for reuse. These difficulties result in increased costs associated with the use of such enzymes due to the necessity for their frequent replacement. Moreover, while free (unimmobilized) enzymes can be used efficiently in batch-type processes, they do not lend themselves to use in continuous, industrial-scale processes.
To reduce the high cost of enzyme replacement, various methods have been devised for immobilizing enzymes prior to their use. Such immobilization permits the enzymes to be conviently removed from the reaction medium for subsequent reuse. These immobilized enzymes may be employed in various reactor systems, such as in packed columns and stirred tank reactors, depending on the nature of the substrate which is being biocatalytically reacted.
Methods proposed for immobilization of an enzyme include the use of a carrier in the form of a solid support made from inorganic or organic material. Such materials include, for example, gamma-alumina, titania, activated granular carbon, granular diatomaceous earth, glass beads, porous glass, pumice-stone, silica gel, metal oxide and aluminium oxide. A compound, or a mixture of compounds, is used to attach the enzyme to this carrier, with polyethylenimine and glutaraldehyde in particular being cited. However, such methods can be disadvantageous in that the enzyme is not tightly-held (by either being bonded thereto or being entrapped therein) to the carrier. Thus, the enzyme can become "detached" (unbonded) from the carrier becoming "free" in the reaction medium. In fact, the forces which exist between the enzyme and the carrier so as to hold them together are often quite weak, such that the enzyme is readily desorbed from the carrier in the presence of the substrate being processed, and lost in the reaction medium.
U.S. Pat. No. 4,713,333 discloses a process wherein enzymes are immobilized on granular diatomaceous earth. That process involves contacting porous granular diatomaceous earth with a solution of polyethylenimine. Then, the diatomaceous earth containing the polyethylenimine is contacted with glutaraldehyde. Finally, an aqueous solution of the enzyme is then added thereto, whereby the enzyme is immobilized thereon. While being particularly useful, immobilized enzymes conjugates formed in this manner can nonetheless still be improved in view of their stability and half-life.
Thus, it can be seen that there remains a need for a method for immobilizing enzymes in an enzyme conjugate, so that the enzyme is tightly-held (or maintained) within the conjugate formed thereby, whereby the immobilized enzyme cannot become "detached" from the remainder of the conjugate and lost in the reaction medium. It can further be seen that there remains a need for immobilized enzyme conjugates in which the enzyme is tightly-held thereto and/or therein (stable), so that during the use of such conjugates, the enzyme loss therefrom to the reaction medium is reduced, whereby a significant improvement in the productivity thereof is realized.